Deparaffinization compositions and methods for their use

ABSTRACT

Compositions and methods are provided for dewaxing wax-embedded biological specimens prior to histochemical analysis. The compositions and methods provided can effectively remove wax or improved wax-based embedding materials, particularly paraffin-based, from specimens during preparation for histochemical or other diagnostic analyses, while minimizing danger to users, achieving compatibility with automated use, and maintaining compatibility with downstream histochemical analyses, particularly immunostaining. Compositions of the invention comprise a paraffin-solubilizing organic solvent, a polar organic solvent, and a surfactant. Compositions can further comprise water. The method involves contacting a wax-embedded specimen with the dewaxing composition to solubilize the wax impregnating the specimen prior to histochemical analysis. The method can comprise the further step of washing the dewaxed specimen immediately after dewaxing with an aqueous wash composition comprising a detergent to remove residual dewaxing composition. Also provided is a kit for dewaxing a wax-embedded specimen, which comprises a dewaxing composition and can further comprise a second composition of (1) an immunostaining reagent or (2) an aqueous wash solution comprising a detergent for removing residual dewaxing solution.

INTRODUCTION

[0001] 1. Technical Field

[0002] This application relates to compositions and methods for removalof wax from wax-embedded biological samples.

[0003] 2. Background

[0004] Paraffin has been used for many years as an embedding medium inthe preparation of tissue specimens for sectioning in a microtome toproduce specimen sections for histological studies. Such embeddingprocesses generally include the well known steps of specimen fixation,dehydration, clearing, paraffin infiltration or impregnation, blockingor embedding in a block of paraffin, slicing the block and specimen intothin sections, mounting the sections on slides, removing the paraffinand solvents employed for this purpose (deparaffinizing), and stainingthe sections prior to microscopic analysis. The primary purpose of theembedding medium is to permit the specimens to be sectioned and mountedin the natural state. Plastic resins have also been used as embeddingmedium to provide a harder specimen that allows cutting of thinnersections. However, the use of paraffin-embedding has the advantage thatthe wax can be dissolved away from specimens prior to staining, allowingsections to be stained in the form of naked slabs of biopolymer andavoiding the extra difficulties and artifacts associated with thepresence of unremovable resin-embedding medium (Horobin 1991).

[0005] Recent improvements in paraffin-embedding compositions broadenits applicability while maintaining its compatibility with downstreammanipulation and analysis of samples. For example, an improvedparaffin-based embedding material, which includes a mixture of paraffinand an effective amount of ethylene-vinyl acetate copolymer (0.5% to 5%by weight of paraffin) allows shorter infiltration time and thinnersections (U.S. Pat. No. 4,497,792). Another improvement, thedouble-embedding technique, yields sections of thin tissue membranes,such as rodent mesenteric membranes that usually measure only 10 micronsin thickness. In this method several membranes are fixed and mounted onfour needles located at the bottom of a plastic box and then embedded inagarose. The agarose block is removed, dehydrated in alcohol, clearedwith HistoPetrol (tradename for a mixture of isoparaffin hydrocarbons),permeated with paraffin and sectioned. The observed tissue morphology iscomparable to that obtained with methacrylate plastic embedding but isless time-consuming, less hazardous since no plastic hardener andactivator are used, and makes immunohistochemical studies easier(Ghassemifar et al. 1992).

[0006] Consequently, deparaffinization of fixed, e.g. formalin fixed,paraffin embedded tissue sections is still a widely used methodology,particularly in hospital histopathology laboratories forimmunodiagnostic purposes.

[0007] Xylene, which is a flammable, volatile and toxic organic solvent,is currently commonly used in protocols to solubilize paraffin fordeparaffinization of specimen sections. Typically, the microscopeslide-mounted specimen is immersed in a xylene bath until the paraffinis solubilized. The deparaffinized specimen is then washed with a seriesof alcohol solutions of decreasing alcohol concentration, typically asbaths in which the specimen is immersed, to remove xylene before a finalwash with water. Efforts have been made to replace xylene in thedeparaffinization process with less toxic and less volatile solvents.Terpene oil (e.g. available under the tradename AmeriClear from BaxterHealth Care Diagnostics, Inc. McGaw Park, Ill.) and isoparaffinichydrocarbons (e.g. available under the tradename Micro-Clear from MicronDiagnostics, Inc., Fairfax, Va.) produced equal deparaffinizationcompared to xylene (Jones et al. 1993). However, a series of alcoholwashes were still required to remove either solvent prior to the waterwash to achieve compatibility with most types of staining, particularlyimmunohistochemical staining. Furthermore, the use of paraffin-embeddedspecimens with automated systems, such as immunostainers, is increasing.

[0008] Accordingly, there is still a need for deparaffinizationcompositions and methods that can effectively remove paraffin orimproved paraffin-based embedding materials from specimens prior tohistochemical or other diagnostic analyses, while minimizing danger tousers, allowing compatibility with automated systems, and maintainingcompatibility with downstream analyses. Deparaffinization compositionsand methods that entail no or limited toxicity or carcinogenicity,produce no or minimal odors, reduce the quantity of toxic solvents used,minimize hazardous wastes, and/or decrease corrosiveness andflammability are needed.

[0009] Cited Literature

[0010] 1. Horobin, R. W., In Histochemical and ImmunochemicalTechniques: Application to pharmacology and toxicology, (1991) Bach, P.and Baker, J., eds., Chapman & Hall, New York, N.Y. pp 1-9.

[0011] 2. Ghassemifar, R. et al. (1992) “A double-embedding techniquefor thin tissue membranes” Biotech. Histochem. 67:363-366.

[0012] 3. Jones, R. T. et al. (1993) “Comparison of deparaffinizationagents for an automated immunostainer” J. Histotechnology 16:367-369.

[0013] 4. Mullin, L. S. et al. (1990) “Toxicology update isoparaffinichydrocarbons: a summary of physical properties, toxicity studies andhuman exposure data” J. Appl. Toxicol. 10:135-142.

SUMMARY OF THE INVENTION

[0014] Compositions and methods are provided for dewaxing wax-embeddedbiological specimens prior to histochemical or other analyses. Thedewaxing compositions and methods provided can effectively remove wax ormodified wax-based embedding materials, particularly paraffin orparaffin-based, from specimens prior to histochemical or other analyses,while minimizing danger to users, allowing compatibility with automateduse, and maintaining compatibility with downstream analyses. Dewaxingcompositions and methods that entail no or limited toxicity orcarcinogenicity, produce no or minimal toxic odors, reduce the quantityof toxic solvents used, minimize hazardous wastes, and/or decreasecorrosiveness and flammability are provided. The compositions andmethods are especially useful for eliminating the use of xylene and forreducing the use of alcohol in preparation of tissue sections forimmunohistochemical staining, particularly in hospital laboratories.Dewaxing compositions of the invention comprise a paraffin-solubilizingorganic solvent, a polar organic solvent, and a surfactant as specifiedbelow in detail. Compositions can optionally comprise water.

[0015] A method for dewaxing biological specimens prior to histochemicalor other analyses is provided. The method involves contacting awax-embedded specimen with a dewaxing composition of the invention tosolubilize the wax impregnating the specimen prior to histochemicalanalysis, such as immunohistochemical staining.

[0016] It is an object of the invention to eliminate the need foralcohol and alcohol baths for post-dewaxing washes by providing a methodwhich involves contacting a dewaxed specimen, which has been dewaxed bya dewaxing composition of the invention, with an aqueous wash solutioncomprising a detergent to remove residual dewaxing composition.

[0017] A kit for dewaxing a wax-embedded specimen is provided. The kitcomprises a dewaxing composition of the invention and can furthercomprise a second composition of (1) an immunostaining reagent or (2) anaqueous wash solution comprising a detergent for removing residualdewaxing solution.

[0018] The present invention eliminates or minimizes the use of toxicorganic solvents in immunohistological laboratories. The compositionsand methodology described herein effectively remove paraffin or otherwax residue from tissue sections and have no adverse effect on thequality of tissue sections prepared for immunohistochemistry.Application of this dewaxing methodology can be extended to otherapplications where removal of wax from tissue sections are desired, suchas in situ hybridization.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0019] The present invention provides new dewaxing solvent compositionsfor removal of paraffin or other waxes from wax-embedded biologicalspecimens for histochemical or other analyses. The compositions comprisea paraffin-solubilizing organic solvent, a polar organic solvent, and asurfactant. In further embodiments the compositions of the invention maybe optionally diluted with water.

[0020] By “wax” is meant a composition used in the histochemical art forembedding biological specimens for histochemical or other analyses thatis solid at room temperature, usually consists of a complex mixture ofhigher hydrocarbons often including esters of higher fatty acids andhigher glycols, may be mineral, natural or synthetic in origin, isharder and more brittle than fats, is soluble in oils and fats, and canoptionally contain additives that enhance its specimen-embeddingproperties. Paraffin is an example of a mineral wax most commonly usedin the histochemical field. Paraffin is typically prepared bydistillation of petroleum, and is a mixture of primarily solid saturatedhydrocarbons.

[0021] By “histochemical” is meant to include the techniques and methodsknown as immunohistochemical, cytochemical, histopathlogic, enzymehistochemical, special stains, microtechnique, in situ hybridization,and the use of molecular probes. Texts illustrating histochemicaltechniques include “Histochemical and Immunochemical Techniques:Application to pharmacology and toxicology,” (1991) Bach, P. and Baker,J., eds., Chapman & Hall, New York, N.Y. pp 1-9, and in “Stains andCytochemical Methods,” (1993) M. A. Hayat, ed., Plenum Press, New York,N.Y., which are incorporated herein by reference.

[0022] The paraffin-solubilizing organic solvent is a non-polarhydrocarbon or mixture of hydrocarbons (e.g. as from a petroleumdistillate) that has a boiling point well above room temperature,preferably above 110° C., more preferably from about 140° C. to about250° C., that is in liquid phase at the temperatures used with thepresent invention (usually 5° to 50° C.), and that is capable ofdissolving paraffin used for embedding biological specimens. Theparaffin-solubilizing solvent can be a complex mixture of long-chainlinear and branched alkane hydrocarbons containing for example esters offatty acids and higher glycols. The paraffin solubility of the solventat 25° C. is typically at least 0.1 gram paraffin per 1 liter ofsolvent, preferably 0.1 gram per 100 ml of solvent, more preferably 0.1gram per 10 ml of solvent, and most preferably capable of a dissolvingan amount of paraffin equal to about 50% of the solvent solutionsweight. The paraffin-solubilizing solvent is further miscible with apolar organic solvent when used in a composition of the invention.Examples of paraffin-solubilizing organic solvents include aromatichydrocarbons, aliphatic hydrocarbons, terpenes, other oils, andpetroleum distillates. Preferred paraffin-solubilizing organic solventshave little or no toxic effects. Furthermore preferred solvents arethose not classified by the Environmental Protection Agency as hazardouswaste. A preferred paraffin-solubilizing solvent furthermore has a flashpoint higher than about 60° C. which minimizes flammability. A preferredsolvent furthermore lacks toxicity, carcinogenicity, and corrosiveness.An isoparaffinic hydrocarbon is an example of a preferredparaffin-solubilizing solvent, in part because of its lack of toxicity,carcinogenicity, corrosiveness and flammability (Mullin et al. 1990).Preferred isoparaffins are branched aliphatic hydrocarbons with a carbonskeleton length ranging from approximately C10 to C15, or mixturesthereof. One preferred isoparaffin hydrocarbon mixture has a flashpointof about 74° C. Another preferred paraffin-solubilizing solvent is amixture of C₁₀ to C₅₀ branched or linear hydrocarbon chains having adistillation range from a boiling point of 150° C. to about 250° C., andhas the general formula of C_(n) H_((2n±m)) where n=10-50 and m=0-4.Mineral spirits is another preferred paraffin-solubilizing organicsolvent. A preferred terpene is limonene. Other terpenes that can beused include terpins, terpinenes and terpineols. Less preferably thesolvent is an aromatic hydrocarbon solvent such as an alkylbenzene, e.g.xylene, or dialkylbenzene, e.g. toluene. Toluene and xylene are lesspreferred because of their toxicity and rating as hazardous waste.Furthermore, as discussed below, even when xylene or toluene are used inembodiments of the invention, subsequent alcohol washes are eliminatedand replaced with a non-hazardous aqueous wash solution.

[0023] The paraffin-solubilizing organic solvent in the composition istypically from about 25% to about 75% by volume of the dewaxingcomposition. Below the lower percent limit of paraffin-solubilizingorganic solvent the dewaxing capability of the composition issignificantly decreased. Above the upper limit of paraffin-solubilizingsolvent an adverse affect on detergent solubility or water solubilityoccurs, which adversely affects the effectiveness of a subsequentaqueous wash. The upper limit of solvent can be selected among the upperlimit values of 50%, 70%, and 75%, while the lower limit of solvent canbe selected from the lower limit values of 25%, 35% and 40%, to obtain avariety of ranges for embodiments of the invention.

[0024] Because these compositions are typically prepared by combiningcomponents without a precise determination of the final volume of thecomposition or accounting for volume changes upon mixing, thepercentages for each component are qualified with the term “about,” withthe understanding that one skilled in the art would appreciate theimprecision of the values as a consequence of composition preparation;however, preferably percentage values are taken to mean their precisevalue when volume changes upon mixing are accounted for.

[0025] The polar organic solvent serves the purpose of dissolving theparaffin-solubilizing solvent, surfactant and optionally water. Thepolar organic solvent is soluble in water to the extent of at least 1 gper 100 g water, preferably 5 g per 100 g water, more preferably 10 gper 100 g water and most preferably the polar organic solvent ismiscible with water. Polar organic solvents include ketones and loweralcohols, which include polyhydroxy alcohols and glycols, and lowerethers. Preferred alcohols are C₁ to C₅ alcohols. Most preferred areethanol, ethylene glycol, isopropanol, propylene glycol and mixturesthereof. A preferred ketone solvent is typically C₃ to C₅ ketone. Mostpreferred ketone solvents are acetone and methyl ethyl ketone. Preferredethers are C₂ to C₆ ethers. Particularly preferred polar organicsolvents are selected from the group consisting of methanol, ethanol,isopropanol, butanol, tert-butanol, allyl alcohol, acetone, ethyleneglycol and propylene glycol, and a mixture thereof. Acetonitrile anddimethylformamide are less preferred polar organic solvents.Furthermore, the polar organic solvent can be a mixture of polar organicsolvents.

[0026] The polar organic solvent in the composition is typically fromabout 25 % to about 75% by volume of the composition. Preferably theamount is from about 35% to about 70%, more preferably from about 40% toabout 65%, and most preferably from about 50% to about 60%. At whatcombination of components a composition is miscible or separates canreadily be determined from a phase diagram showing phase separation fordifferent relative amounts of the components of the solution/mixture.

[0027] Surfactants include cationic surfactants, anionic surfactants,non-ionic surfactants, and zwitterionic surfactants. A number ofbiological detergents (surfactants) are listed as such by Sigma ChemicalCompany on pages 1502-1508 of its 1991 Catalog of Biochemicals andOrganic Compounds for Research and Diagnostic Agents. The surfactantserves the purpose of a detergent since it has both hydrophilic andhydrophobic properties. A surfactant for use in the invention is solublein the solvent used in a composition of the invention. Preferredsurfactants are detergents that are soluble in water, ethanol andacetone. Most preferred are those that do not substantially interferewith downstream histochemical analyses, which can be determined, forexample, by immunostaining using a solution containing the surfactant.

[0028] Surfactants that can be used in compositions of the inventioninclude cationic surfactants of the formula

[0029] wherein R₁ is methyl, ethyl or propyl or isopropyl where n is 1or 2; R₂ is an alkyl group selected from C₈H₁₇ to C₃₀H₆₁ or benzylgroup; and R₃ is (CH₂)_(m), where m is from 1 to 10, or R₃ is(OCH₂CH₂)_(p) where p is from 1 to 10. Cationic surfactants of thisformula are soluble in the polar organic solvents. Many preferredembodiments of the invention contain the cationic surfactantbenzalkonium chloride or benzethonium chloride. Additional cationicdetergents, not necessarily of this formula, includedodecyltrimethylammonium bromide, benzyldimethylhexadecyl ammoniumchloride, cetylpyridinium chloride, methylbenzethonium chloride, and4-picoline dodecyl sulfate.

[0030] Other surfactants that can be used in the compositions of theinvention include anionic surfactants having the formula

[0031] wherein R₁ is C₆H₁₃ to C₃₀H₆₁, and R₃ is CH₂ or phenyl group.Anionic surfactants of this formula are soluble in polar organicsolvent. Examples of anionic detergents, not necessarily having thisformula, include alginic acid, caprylic acid, cholic acid,1-decanesulfonic acid, deoxycholic acid, 1-dodecanesulfonic acid,N-lauroylsarcosine, and taurocholic acid. Other anionic syntheticnon-soap detergents, which are represented by the water-soluble salts oforganic sulfuric acid reaction products, have in their molecularstructure an alkyl radical containing from about 8 to 22 carbon atomsand a radical selected from the group consisting of sulfonic acid andsulfuric acid ester radicals. Examples of these are the sodium orpotassium alkyl sulfates, derived from tallow or coconut oil; sodium orpotassium alkyl benzene sulfonates; sodium alkyl glyceryl ethersulfonates; sodium coconut oil fatty acid monoglyceride sulfonates andsulfates; sodium or potassium sales of sulfuric acid esters of thereaction product of one mole of a higher fatty alcohol and about 1 to 6moles of ethylene oxide per molecule and in which the alkyl radicalscontain from 8 to 12 carbon atoms; the reaction product of fatty acidsesterified with isethionic acid and neutralized with sodium hydroxide,sodium or potassium salts of fatty acid amide of a methyl tauride; andsodium and potassium salts of SO₃-sulfonated C₁₀-C₂₄ α-olefins.

[0032] Further surfactants that can be used in compositions of theinvention include non-ionic surfactants having the formula

[0033] wherein R is a linear or branched C1 to C10 alkyl group and X isan integer from 5 to 40. Most preferably R is

[0034] Non-ionic surfactants of this formula are soluble in polarorganic solvents. Examples of nonionic detergents, not necessarilyhaving this formula, include decanoyl-N-methylglucamide, diethyleneglycol monopentyl ether, n-dodecyl β-D-glucopyranoside, polyoxyethyleneethers of fatty acids (particularly C₁₂-C₂₀ fatty acids, (e.g., soldunder the trade name Triton), ethylene oxide condensates of fattyalcohols e.g. sold under the name Lubrol), polyoxyethylene sorbitanfatty acid ethers (e.g., sold under the trade name Tween), and sorbitanfatty acid ethers (e.g., sold under the trade name Span). Nonionicsynthetic detergents made by the condensation of alkylene oxide groupswith an organic hydrophobic compound. Typical hydrophobic groups includecondensation products of propylene oxide with propylene glycol, alkylphenols, condensation product of propylene oxide and ethylene diamine,aliphatic alcohols having 8 to 22 carbon atoms, and amides of fattyacids. Also nonionic detergents such as amine oxides, phosphine oxidesand sulfoxides having semipolar characteristics and be removed. Specificexamples of long chain tertiary amine oxides includedimethyldodecylamine oxide and bis-(2-hydroxyethyl)dodecylamine.Specific examples of phosphine oxides are found in U.S. Pat. No.3,304,263 which issued Feb. 14, 1967, and includedimethyldodecylphosphine oxide and dimethyl-(2-hydroxydodecyl) phosphineoxide. A preferred non-ionic detergent surfactant is Triton X-100, whichis a tradename for a polyoxyethylene ether of fatty acids (particularlyC₁₂-C₂₀ fatty acids).

[0035] Zwitterionic surfactants include known compounds of the formulaN-alkyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate. Examples ofzwitterionic detergents include3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (commonlyabbreviated CHAPS),3-[(cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate(generally abbreviated CHAPSO),N-dodecyl-N-dimethyl-3-ammonio-1-propanesulfonate, andlyso-α-phosphatidyl-choline.

[0036] The surfactant in the composition is typically from about 0.5% toabout 20% weight to volume (g/100 ml) of the composition. Below thelower limit of surfactant poor solubility of wax in the composition isobserved. The upper limit of surfactant is a factor of the surfactant'ssolubility limit. The amount of surfactant is preferably from 0.5% toabout 15% by weight, more preferably from about 0.5% to about 10%surfactant by weight, most preferably from about 0.5% to about 5% byweight.

[0037] Compositions of the invention can also contain water. Mostpreferably the water in a composition is a saturating amount of water.Above this upper limit phase separation of the composition occurs.Typically water is less than or about 10% by volume of the composition.Some embodiments of the invention, for example as exemplified in theExamples, have less than about 7% water, some have from about 0.5% toabout 1.5% water, and still others have less than about 1% water byvolume.

[0038] In some embodiments of the invention, the dewaxing compositionsor the aqueous wash solutions contain buffer, salts or other reagentsuseful for wax-solubilization, washes, or subsequent histochemicalsteps, so long as such optional reagents do not interfere with thewax-solubilizing capability of the composition, the efficiency of awashing step, or subsequent histochemical steps. Reagents useful forsubsequent processing or histochemical steps include carboxylic acidesters, enzymes such as lipases, and nucleophilic reagents as describedin co-owned U.S. application Ser. No. 07/821,931, filed Jan. 16, 1992,entitled “Enhancement of immunochemical staining in aldehyde-fixedtissues,” which is incorporated herein by reference. Optional agents canserve to expose or enhance aldehyde-fixed tissue antigen(s) forimmunohistochemical staining. Additional optional reagents includeanti-microbial agents and stabilizers that increase composition shelflife. Such anti-microbial agents and stabilizers are well known in thefield. Such reagents are typically used at extremely small percentages,typically below 0.1%, compared to the main components. Preferredreagents are those that do not interfere with downstream histochemicalanalyses.

[0039] Each of the individual components of the compositions of theinvention is either commercially obtainable, is isolated from naturalsources using known procedures, or is synthesized according to knownprocedures.

[0040] Compositions of the invention are prepared by simple mixing ofthe components in the indicated amounts.

[0041] Methods of preparing biological samples for sectioning via wax-or paraffin-impregnation are generally well known and easily carriedout. The process is quite simple and involves contacting a wax-embeddedspecimen with a dewaxing composition of the invention to solubilize thewax that impregnates the specimen prior to histochemical analyses, suchas immunostaining. The method optionally comprises a further step ofcontacting the dewaxed specimen immediately after dewaxing with anaqueous washing composition comprising a detergent to remove residualdewaxing composition.

[0042] Although the dewaxing process is typically and convenientlycarried out at room temperature, without the need for a temperaturecontrolled bath, a more precise control of the required time forsatisfactory dewaxing and washing is available if temperature-controlledbaths are used. Heating decreases processing time. Operable temperaturesrange from 5° to 50°, preferably from about 15° C. to about 45° C., andmore preferably from about 25° C. to about 40° C.

[0043] Typically the wax-embedded specimen is contacted with acomposition of the invention for a time sufficient to solubilize all orpart of the wax embedding the specimen. Factors influencing thesolubilization time include temperature, thickness of the specimensection, and wax composition. Time for any particular specimen type isbest determined empirically. However, five minutes of contact is usuallysufficient for specimens mounted on microscope slides. A sectionedspecimen, typically affixed to a microscope slide, is contacted with acomposition of the invention in any number of ways. Preferably, thespecimen is immersed in a bath containing the composition, oralternatively an amount of composition sufficient to solubilize the waxcan be placed on the specimen such that the specimen is covered by thecomposition. After sufficient time of contact has elapsed for waxsolubilization to occur, the specimen is removed from contact with thecomposition, and excess composition is removed from the specimen, forexample by draining or blotting. Optionally, a second or even a furtherdeparaffinization step or steps are performed, preferably with freshdewaxing composition, to further assure removal of wax from thespecimen.

[0044] The invention decreases or eliminates the requirement of alcoholbaths for post-dewaxing washes. Post dewaxing washes are not alwaysrequired with compositions of the invention. If such a step provesdesirable (because of a particularly sensitive immunostaining procedure,for example) the dewaxed specimen can be contacted with an aqueous washcomposition of the invention which comprises a detergent. A preferredwash solution comprises a buffer and a detergent. Preferably thedetergent is non-ionic. A preferred buffer/detergent wash solution isphosphate buffered saline with about 1% nonionic surfactantpolyoxyethylene ether such as BRIJ-35 (tradename for the nonionicsurfactant polyoxyethyleneglycol dodecyl ether or polyoxyethylene (23)lauryl ether). Typically the amount of detergent is from about 0.1% toabout 5% (weight to volume), preferably from about 0.1% to 2%, and mostpreferably about 1%. The pH of the wash composition is most preferablyneutral to avoid adversely affecting downstream histochemical,particularly immunochemical, analyses. The pH can range from about 4 toabout 10, preferably from about 5 to about 8, more preferably from about7.2 to about 7.6, and most preferably 7.4 to about 7.5. A preferredbuffer is one which does not interfere with downstream analyses and/orcan be readily removed with a subsequent aqueous wash or optional waterwash. Phosphate buffered saline or Tris containing buffers are examplesof preferred buffers. Washing can occur in any number of ways, includingimmersion in a wash bath, flowing wash solution over the specimen, ordiffusing or permeating the wash solution throughout the specimen. Washtime is best determined empirically; however, five minutes is usuallysufficient. Multiple rinses and larger amounts of washing solution canbe used to achieve increased removal of deparaffinization solution. Asingle wash is sufficient for most purposes; however, a second wash ispreferred if removal is not sufficient. Optionally, the specimen isfinally washed or rinsed in water. A water wash of 3 minutes is usuallysufficient for the most rigorous conditions. After washing the specimenis then ready for histochemical or other analyses.

[0045] The compositions of the invention, including the wash solutions,are compatible with automated staining systems, as described, forexample, in co-owned application U.S. Ser. No. 08/129,243, which ishereby incorporated by reference. Dewaxed slides can be provided to anautomated stainer or an automated stainer can be provided withcompositions of the invention to allow automated dewaxing of the slidesprior to automated analyses.

[0046] Although preferred surfactants and other components used in adewaxing solution of the invention are those that do not typicallyinterfere with downstream analyses, particularly at the residual levelsremaining on the specimen after the wash procedures, methods known inthe art may be applied to enhance surfactant (or other component)removal should residual surfactant (or other component) cause problemsin downstream analyses. For residual surfactant removal solublecompounds known to bind a surfactant may be included in an aqueous washsolution. For example, cyclodextrins are known to bind certainsurfactants (U.S. Pat. No. 5,032,503) and may be included in a washsolution. Protein, such as bovine serum albumin, can be included in awash solution to bind and remove residual surfactant. In one preferredembodiment, a surfactant that does not interfere with the downstreamanalyses, but that can displace the residual surfactant, can be used inan aqueous wash solution. This displacing surfactant is preferablyeasily removed with a water wash. Polyoxyethylene alkyl ether typenon-ionic surfactants are a preferred wash surfactant. BRIJ-35(tradename for polyoxyethyleneglycol dodecyl ether) is an example of onesuch surfactant.

[0047] Also provided is a kit for dewaxing a wax-embedded specimen. Thekit comprises a container of dewaxing composition and can furthercomprise a second container of (1) an histochemical staining reagent or(2) an aqueous wash solution of the invention comprising a detergent forremoving residual dewaxing solution. The containers are typicallylocated in a receptacle specifically adapted to hold them. Preferablythe wash solution contains a buffer and a detergent. In one embodimentthe histochemical staining reagent is an immunostaining reagent. Inanother embodiment the histochemical staining reagent is an in situhybridization reagent. The kit can be a component of a larger kit forhistochemical analyses, such as in a kit for use with automatedimmunostainers. Any of the other reagents described herein can be usedin the kit in combination with the specified components.

[0048] The compositions and methods of the invention are suitable foruse in a variety of histochemical applications, particularlyimmunochemical staining. In situ hybridization with nucleic acid probesis another particularly pertinent use compatible with compositions andmethods of the invention.

[0049] The present invention eliminates or minimizes the use of certaintoxic organic solvents in immunohistological laboratories. Thecompositions and methodology described herein effectively removesparaffin and other waxes residues from tissue sections and has noadverse effect on quality of tissue sections prepared forimmunohistochemistry. Application of this deparaffinization methodologycan be extended to other applications where removal of paraffin andother waxes from tissue sections are necessary. In preferred embodimentsusing isoparaffins, the compositions have a very low order of acutetoxicity, being practically non-toxic by oral, dermal and inhalationroutes. In addition the compositions allow a method of deparaffinizationthat eliminates the use of graded alcohol washes. Accordingly, theembodiments of the present invention meet the need of providingcompositions and methods that minimize dangers to the user and minimizethe creation of hazardous waste.

[0050] The invention now being generally described, the same will bebetter understood by reference to the following detailed examples whichare provided for illustration and are not to be considered as limitingthe invention unless so specified.

EXAMPLES Example 1 Deparaffinizing Compositions

[0051] The following examples of deparaffinization compositions arepresented by way of illustration of embodiments of the invention and notintended to limit the invention.

[0052] Composition 1 was prepared by mixing reagent alcohol (275 ml; apremixed solution of 90% v/v anhydrous ethyl alcohol, 5% v/v methylalcohol and 5% v/v isopropyl alcohol), limonene (100 ml), water (25 ml)and benzalkonium (20 g).

[0053] Composition 2 was prepared by mixing reagent alcohol (100 ml),limonene (100 ml) and benzalkonium (15 g).

[0054] Composition 3 was prepared by mixing reagent alcohol (100 ml),isoparaffinic hydrocarbon (100 ml) and benzalkonium (15 g). Theisoparaffinic hydrocarbon solvent used in the compositions of thisexample is available as Micro-Clear, a tradename of Micron Diagnostics,Inc., for their isopamffinic hydrocarbon solvent.

[0055] Composition 4 was prepared by mixing reagent alcohol (50 ml),limonene (50 ml), water (0.6 ml) and benzalkonium (10 g).

[0056] Composition 5 was prepared by mixing reagent alcohol (50 ml),iso-paraffin (50 ml), water (0.6 ml) and benzalkonium (15 g).

[0057] Composition 6 was prepared by mixing reagent alcohol (100 ml),iso-paraffin (50 ml), mineral spirits (50 ml) and benzalkonium (15 g).

[0058] Composition 7 was prepared by mixing reagent alcohol (50 ml),iso-paraffin (50 ml), water (0.9 ml) and Triton-X100 (10 g).

[0059] Composition 8 was prepared by mixing reagent alcohol (65 ml),iso-paraffin (45 ml) water (0.5 ml) and BRIJ-35 (1.0 g).

[0060] Composition 9 was prepared by mixing reagent alcohol (54 ml),isoparaffin (35 ml) water (1.0 ml) and BRIJ-35 (2.5 g).

Example 2 Deparaffinization

[0061] Deparaffinization of slide mounted tissue specimens usingCompositions 1-9 of Example 1 was performed prior to immunohistologicalanalyses. Human tissues used in this study included skin, pancreas,tonsil, spleen, lung, breast, prostate, colon carcinoma, melanoma andastrocytoma. Compositions 1-9 were tested individually. Each 4 micronparaffin embedded tissue section on a slide was covered with 1.9 ml of adeparaffinization Composition 1-9. After 5 minutes, thedeparaffinization composition was removed and 1.0 ml of freshdeparaffinization composition was added to cover the section for anadditional 5 minutes. A third deparaffinization of the section for anadditional 5 minute in fresh deparaffinization composition was alsocarried out. Immediately after the final deparaffinization, slides wererinsed in an aqueous wash composition of PBS with 1% BRIJ-35 for 5minutes. The slides were then rinsed in tap water for 3 minutes and usedfor immunohistochemistry.

Example 3 Deparaffinization

[0062] In this Example a second method for deparaffinization of slidemounted tissue specimens using Compositions 1-9 of Example 1 wasperformed prior to immunohistological analyses. Human tissues used inthis study included skin, pancreas, tonsil, spleen, lung, breast,prostate, colon carcinoma, melanoma and astrocytoma. Compositions 1-9were tested individually. Each slide containing paraffin embedded tissuesections was immersed in a glass-jar containing 60 ml of one of thedeparaffinization Compositions 1-9. After 5 minutes, thedeparaffinization composition was decanted and replaced with freshdeparaffinization composition and the slides were deparaffinized for anadditional 5 minutes. A third five-minute deparaffinization was alsocarried out. Immediately after deparaffinization, slides were rinsed inan aqueous wash composition containing PBS with 1% BRIJ-35 for 5minutes, rinsed in tap water for 3 minutes, and used forimmunohistochemistry.

Example 4 Deparaffinization in Xylene

[0063] A widely used, standard deparaffinization protocol involvingxylene was performed as a control. Slides containing paraffin embeddedtissue sections were immersed in 100% xylene for 5 minutes followed bytwo changes in fresh 100% xylene for 5 minutes each. Afterwards, theslides were immersed in a bath of 100% alcohol twice for three minuteseach time. The slides were then immersed sequentially in baths of 95%alcohol, 85% alcohol and then 75% alcohol for three minutes in eachbath. The slides were finally rinsed in tap water for 3 minutes and usedfor immunohistochemistry. A series of slides were prepared followingthis protocol but substituting either limonene or Micro-Clear forxylene.

Example 5 Effectiveness of Paraffin Removal

[0064] The deparaffinized slides prepared as in Examples 2, 3, and 4were examined for effectiveness of paraffin removal. After the slideswere deparaffinized with deparaffinization Compositions 1-9 and washedaqueous wash composition of PBS with 1% BRIJ-35, no paraffin residue wasleft on the sections or other location of the slides. No paraffinresidue was detected on sections or slides deparaffinized with thecontrol procedure using xylene and hydrated with graded alcohols andwater. There was no discernible difference in effectiveness for paraffinremoval among xylene, limonene, isoparaffin, and deparaffinizationCompositions 1-9 of Example 1.

Example 6 Effect of Deparaffinization Solvents on ImmunohistochemistryStaining

[0065] Normal or tumorous human tissues including skin, pancreas,tonsil, spleen, lung, breast, prostate, colon carcinoma, melanoma andastrocytoma, were stained with corresponding monoclonal antibodies todetermine effects of deparaffinization compositions onimmunohistochemical staining. Xylene deparaffinized tissue sectionslides were used as standard controls. Slides containing sectionsdeparaffinized as described in Examples 2, 3 and 4 were examined forcompatibility to immunohistological analyses. Deparaffinized slides wereimmersed in Block Solution I (a tradename of BioGenex, San Ramon,Calif., for a solution of PBS and 3% hydrogen peroxide), for 10 minutes.Each slide was then rinsed in PBS twice, 5 minutes each time. Twohundred microliters of primary antibodies (obtained from BioGenex underthe tradename Ready to Use Antibodies) were incubated with theirrespective sections for 30 minutes or 2 hours according to individualstaining protocols provided by the supplier. The following monoclonalantibodies were used in immunohistochemistry: anti-human cytokeratincocktail, anti-NSE, anti-insulin, anti-LCA, anti-kappa chain,anti-Q-band, anti-L26, anti-factor VIII, anti-CEA, anti-p53,anti-Cerb-2, anti-PR, anti-vimentin, anti-PSA, anti-HMB45, anti-S-100and anti-GFAP. The slides were then washed in PBS three time, 5 minuteseach. After a 20 minute incubation with biotinylated secondaryantibodies (available under the tradname Super Sensitive Link fromBioGenex), the slides were washed in PBS three times, 5 minutes eachtime. Slides were then incubated with a stock solution ofperoxidase-conjugated streptavidin (available under the tradename SuperSensitive Label from BioGenex) for 20 minutes and washed three times inPBS. The chromogenic reaction was carried out using AEC(3-amino-9-ethylcarbozole) for peroxidase and Fast Red for alkalinephosphatase. After color development, each slide was rinsed in tapwater, counter-stained, mounted and examined by light microscopy.

[0066] Intensity of immunostaining reactivity was evaluated by alight-microscope. There was no difference in immunostaining intensityamong slides deparaffinized with deparaffinization Compositions 1-9 andthe control deparaffinized with xylene.

Example 7 Effect of Frequency of Solvent Composition Changes onDeparaffinization

[0067] Composition 8 was used to evaluate the effect of frequency ofcomposition changes on deparaffinization. The extent of paraffin removalwas determined by visual inspection of the treated slides. In thepresence of residual paraffin water formed numerous, small droplets onthe slide's surface, which had a waxy-appearance. Upon completedeparaffinization water formed an even film on the slide surface. Afterslides were immersed in Composition 8 for five minutes and then washedwith aqueous wash buffer: PBS containing 1 % BRIJ-35, there was residualparaffin left on the slides. There was no discernable residual paraffinleft on the slides when specimens were deparaffinized by immersion inComposition 8 for five minutes, followed by immersion in a freshsolution of Composition 8 for five minutes, and finally washed with theaqueous wash solution. Specimens were also deparaffinized by a protocolof two changes of Composition 8 followed by the aqueous wash. There wasno difference in terms of intensity of immunohistochemical stainingamong the three deparaffinization conditions.

[0068] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0069] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A composition, comprising a paraffin-solubilizingorganic solvent from about 25% to about 75% by volume of saidcomposition, a polar organic solvent from about 25% to about 75% byvolume of said composition, and a surfactant from about 0.5% to about20% by weight to volume of said composition.
 2. The composition of claim1, which further comprises water at less than or about 10% by volume. 3.The composition of claim 1, wherein the paraffin-solubilizing organicsolvent comprises an aromatic hydrocarbon, a terpene or isoparaffinichydrocarbon.
 4. The composition of claim 3, wherein the terpene islimonene.
 5. The composition of claim 3, wherein theparaffin-solubilizing organic solvent comprises an isoparaffinichydrocarbon.
 6. The composition of claim 1, wherein the polar organicsolvent is selected from the group consisting of alcohols, ketones, andethers.
 7. The composition of claim 6, wherein the polar organic solventcomprises a solvent or a mixture of solvents selected from the groupconsisting of methanol, ethanol, isopropanol, butanol, acetone, ethyleneglycol, and propylene glycol.
 8. The composition of claim 1, wherein thesurfactant is present at about 0.5% to about 15% weight to volume. 9.The composition of claim 6, wherein the alcohol is methanol, ethanol,propanol, isopropanol, tert-butanol or allyl alcohol.
 10. Thecomposition of claim 3, wherein the aromatic hydrocarbon solvent is analkylbenzene or dialkylbenzene.
 11. The composition of claim 1, whereinthe surfactant comprises a cationic or anionic surfactant.
 12. Thecomposition of claim 1, wherein the surfactant is a non-ionicsurfactant.
 13. The composition of claim 11, wherein the cationicsurfactant has the formula

wherein R₁ is methyl, ethyl or propyl or isopropyl where n is 1 or 2; R₂is an alkyl from C₈H₁₇ to C₃₀H61 or a benzyl group; and R₃ is (CH₂)_(m),where m is from 1 to 10, or R₃ is (OCH₂CH₂)_(p) where p is from 1 to 10.14. The composition of claim 13, wherein the cationic surfactant isbenzalkonium chloride or benzethonium chloride.
 15. The composition ofclaim 11, wherein the anionic surfactant has the formula

wherein R₁ is C₆H₁₃ to C₃₀H₆₁, and R₃ is CH₂ or phenyl group.
 16. Thecomposition of claim 12, wherein the non-ionic detergent surfactant hasthe formula

wherein R is a linear or branched C1 to C10 alkyl group and X is from 5to
 40. 17. The composition of claim 16, wherein the non-ionic surfactantcontains polyoxyethylene ethers of C₁₂ to C₂₀ fatty acids.
 18. A processfor removing wax from a wax-embedded specimen, the process comprisingcontacting the wax-embedded specimen with a composition comprising aparaffin-solubilizing organic solvent, a polar organic solvent, and asurfactant to solubilize the wax in specimen.
 19. The process of claim18, which further comprises the step of washing the dewaxed specimenimmediately after dewaxing with an aqueous wash solution comprising adetergent.
 20. A kit for use in dewaxing a wax-embedded specimen, thekit comprising: a receptacle adapted to hold individual reagentcontainers, a first container containing a dewaxing compositioncomprising a paraffin-solubilizing organic solvent, a polar organicsolvent, and a surfactant to solubilize the wax in the specimen, and asecond container containing (1) an immunostaining reagent or (2) anaqueous wash solution comprising a detergent for removing residualdewaxing solution.